viral covert mortality disease (vcmd): disease card

@NACA, May 2020

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Diseases of Crustaceans ─ Viral Covert Mortality Disease (VCMD)

Signs of Disease

Important: affected animals may show one or

more of the signs below, but the infection may be

present in the absence of any signs, especially

during the early phase of infection.

Disease signs at pond level (Level I

diagnosis)

• Most of moribund shrimp stay at the bottom

and die; moribund and dead shrimp can be

observed daily;

• High mortality follows a rapid change in water

temperature, especially at above 28oC.

Disease signs at animal level (Level I

diagnosis)

The following can be observed in infected

shrimps:

• Hepatopancreatic atrophy and necrosis

(Figures 1 and 2);

• Empty stomach and gut;

• Soft shell;

• Slow growth;

• In many cases, abdominal muscle whitening

and necrosis (Figures 1 and 2).

Figure 1. VCMD in cultured white shrimp (Penaeus

vannamei). White arrows indicate atrophy and a faded

colour to the hepatopancreas. Black arrows show whitening

of abdominal muscle segments.

Source: QL Zhang

Figure 2. VCMD in experimentally infected whiteleg shrimp (P.

vannamei). White arrow indicates atrophy and color fading of the

hepatopancreas compared to the normal shrimp with dark

heaptopancreas (white arrow head).

Source: QL Zhang

Disease Agent

VCMD is caused by covert mortality nodavirus

(CMNV), a positive single strand RNA virus that

has been classified in the family Nodaviridae.

Similar Diseases

• Infection with Infectious myonecrosis virus (IMNV) • White tail disease (Infection with Penaeus vannamei nodavirus)

@NACA, May 2020



Viral Covert Mortality Disease

Host Range

Crustaceans known to be susceptible to infection with covert mortality nodavirus (RT-PCR and ISH

positive) include Penaeus vannamei, P. chinensis, P. japonicus, P. monodon, Macrobrachium

rosenbergii, Procambarus clarkii, Exopalaemon carinicauda, Ocypode cordimanus, Diogenes edwardsii,

Corophium sinense, Parathemisto gaudichaud, and Tubuca arcuate (Zhang et al., 2014; 2017a; Li, et al.,

2018a; Liu et al., 2018). Fish species, including Mugilogobius abei, Carassius auratus, and Paralichthys

olivaceus may also be susceptible to the virus, according to the results from ISH (Zhang et al., 2018;

Wang et al., 2018, 2019).

Presence in Asia-Pacific

Shrimp samples collected from China were found positive for CMNV by RT-PCR, ISH (Zhang et al.,

2014; Zhang et al., 2017; Zhang 2019). Shrimp samples collected from Thailand were found positive for

CMNV by RT-PCR and ISH (Pooljun et al., 2016; Thitamadee et al., 2016). Material collected from

Thailand, however, consisted of only non-diseased, grossly normal shrimp. They showed no ISH

reactions in the muscle tissue and the positive ISH reactions were relatively weak and occurred only in

nuclei of the tubule epithelial cells of the hepatopancreas. This differed from the Chinese samples where

the positive signals arose from the cytoplasm of such cells (Fig. 5).

Epidemiology

• Infection with CMNV usually occurs within 30-80 days post stocking, with cumulative mortality up

to 80%. In serious cases, it occurs within 10-20 days of stocking post larvae into grow-out ponds.

Cases of asymptomatic infection detected with CMNV kits were also found in farms.

• Horizontal transmission through cannibalistic behavior of shrimp. • Vertical transmission of CMNV via sperm or oocytes in Exopalaemon carinicauda (Liu et al., 2017).

• Some wild crustaceans in the ponds are vectors of the disease (Liu et al., 2018).

• Migratory birds, aquatic insects and humans are likely mechanical vectors of the disease.

Horizontal Transmission

Eleven species of invertebrates inhabiting shrimp ponds were found to be CMNV positive using RT-

nPCR or RT-LAMP. These include brine shrimp Artemia sinica, barnacle Balanus sp., rotifer

Brachionus urceus, amphipod Corophium sinense, Pacific oyster Crassostrea gigas, hermit crab

Diogenes edwardsii, common clam Meretrix lusoria, ghost crab Ocypode cordimundus, hyperiid

amphipod Parathemisto gaudichaudi, fiddler crab Tubuca arcuata, and an unidentified gammarid

amphipod (Liu et al., 2018). Five wild crustacean species, including Cor. sinense, Dio. edwardsii, Ocy.

cordimanus, Par. gaudichalldi, and Tub. arcuata, were also tested to be ISH positive of CMNV. These

five species might also act as reservoir hosts of CMNV in horizontal transmission of the disease (Liu et

al., 2018).

@NACA, May 2020




Figure 4. A and B: H&E staining and ISH for necrotic muscle of E. carinicauda naturally infected with CMNV. The muscle

showed fragmentation tending towards coagulative and dissolving necrosis (black triangles). The black arrows and open

arrows indicated the karyopyknotic nuclei and the purple hybridization signal of CMNV probe, respectively. Scale bars

=20 μm (A and B). Source: QL Zhang

Figure 5. A and B: H&E staining and ISH for atrophied and necrotic hepatopancreas epithelium of P. monodon naturally

infected with CMNV. The black triangles and open arrows indicated the necrotic epithelium and the purple hybridization

signal of CMNV probe, respectively. Scale bars =20 μm (A and B). Source: QL Zhang

Histological Images (Level II diagnosis)

Figure 3. H&E staining and in situ hybridization (ISH) for necrotic muscle of P. vannamei naturally infected with CMNV. The

muscle showed fragmentation tending towards coagulative and dissolving necrosis (black triangles). The black arrows and

the open arrows indicated the karyopyknotic nuclei and purple hybridization signal of the CMNV RNA probe, respectively.

Scale bars = 20 μm (A), 20 μm (B). Source: QL Zhang

@NACA, May 2020




Molecular Diagnostics (Level III diagnosis)

Reverse transcription amplification based methods

Different molecular diagnostic methods were established based on the sequence of RNA-dependent RNA

polymerase (RdRp). The reported methods include:

• A reverse transcriptase, nested PCR (RT-nPCR) (Zhang, et al., 2014).

• A RT-LAMP method (Zhang et al., 2017b).

• A sensitive TaqMan RT-PCR (Pooljun et al., 2016).

• A high compatibility TaqMan real time RT-PCR (Li et al., 2018b)

Nucleic acid probe based in situ hybridization (ISH) methods

• An ISH method for CMNV was initially described in 2014 (Zhang, et al., 2014).

• An alternative ISH method was published (Zhang, et al., 2017).

There is no sequence overlap between the RT-nPCR and any of the TaqMan RT-PCR method or the RT-

LAMP. Therefore, RT-nPCR results can be verified with any of the TaqMan RT-PCR methods or the

RT-LAMP tests. It is not valid to verify the results between RT-nPCR and ISH, or between the RT-

LAMP and any of TaqMan RT-PCR methods, as there are overlaps over the targeting sequences of the

relevant methods. ISH and RT-nPCR may bring a high risk of contamination to each other. It is

discouraged to run these two tests in the same room or without a diagnostic laboratory Quality

Management System. Due to the lack of proofreading activity of RdRp, CMNV genomes have characteristically high mutation

rates, which bring the risk of false negatives. Touch down PCR combining with sequencing may be

applied to find out false negatives caused by possible mutations.

List of Expert(s):

Dr. Qingli Zhang Maricultural Organism Diseases Control & Molecular Pathology Laboratory

Yellow Sea Fisheries Research Institute

Chinese Academy of Fishery Sciences

106 Nanjing Road

Qingdao, SD 266071

PR China

[email protected]

@NACA, May 2020




Information presented is based on current knowledge and as reviewed by members of NACA’s Asia

Regional Advisory Group on Aquatic Animal Health (AG). As there are many unknowns regarding

VCMD, the disease card will be updated as new information becomes available through peer-reviewed

studies, expert opinions and experiences from primary producers.

Table 1. Primers and probes for the above-mentioned molecular diagnostic methods.

Methods Primers/Probe Sequences (5’-3’) Ta Target

region

RT-nPCR [1] CMNV-7F1 AAATACGGCGATGACG 45°C 619 bp

(232-850) CMNV-7R1 ACGAAGTGCCCACAGAC

CMNV-7F2 CACAACCGAGTCAAACC 50°C 165 bp

(256-420) CMNV-7R2 GCGTAAACAGCGAAGG

RT-LAMP

[2]

F3 TGCCAAGCAAATACGAGCT 65°C 179 bp

(873-1051) B3 CATCAGCGATGTCACGGC

FIP GTCGTCGACGGTTAGGTTGCGTTTTCCAAGCAC

TTCCCGACAA

BIP CGTCCAAAAGGACCTCCGCATTTTTGGAGACCT

TGGTCACGC

LF GCTCACGGCTTTGGATACC

LB GATTGCATGCGTCAACCTCA

TaqMan RT-

PCR [3]

CMNV-F ACCTCCGCAATCTGATTG 60°C 130 bp

(981-1110) CMNV-R GGGTCTACTTTCGTTGGA

CMNV-

TaqMan

FAM-CGCTACCACTGTCGGCTTGT-TAMRA

TaqMan RT-

PCR [4]

CMNV-Taq-F CGAGCTAATCCAAGCACTTC 52.7°C 198 bp

(886-1084) CMNV-Taq-R ACCTGTTAGGTACGCTACCA

CMNV-Taq-P FAM-CGCTCACGGCTTTGGATACCTT-TAMRA

PCR for ISH

probe [5]

CMNV-

7Probe-F

GGCGATGACGGCTTGA By

Labeling

Kit

201 bp

(238-438)

CMNV-

7Probe-R

GGCGGTGAGATGGATTTT

PCR for ISH

probe [6]

CMNV-ISH-F CTACTGCAGAATACGGCGATGACGG By

Labeling

Kit

227 bp

(233-459) CMNV-ISH-R CAGAAGCTTAAAGGGACGGAAGGGTT

[1] Zhang, et al., 2014; [2] Zhang et al., 2017b; [3] Pooljun et al., 2016; [4] Li et al., 2018; [5] Zhang, et al., 2014;

[6] Zhang et al., 2017a.

@NACA, May 2020




References

Li X.P., Wan X.Y., Huang J., Zhang Q.L, Qiu L., Song Z.L., Cheng D.Y. 2018a. Molecular epidemiological survey on covert mortality

nodavirus (CMNV) in cultured crustacean in China in 2016 – 2017. Progress in Fishery Sciences. (In Chinese) DOI: 10.19663/

j.issn2095-9869.20180420003.

Li X.P., Wan X.Y., Xu T.T., Jie Huang., Zhang Q.L. 2018b. Development and validation of a TaqMan RT-qPCR for the detection of

convert mortality nodavirus (CMNV). J Virol Methods. DOI: 10.1016/j.jviromet.2018.10.001

Liu S, Li J.T., Tian Y., Wang C., Li X.P., Xu T.T., Li J. Zhang Q.L., 2017. Experimental vertical transmission of Covert mortality

nodavirus (CMNV) in Exopalaemon carinicauda. J Gen Virol. 98(4):652-661.

Liu S, Wang X.H., Xu T.T., Li X.P., Du L.C., Zhang Q.L., 2018. Vectors and reservoir hosts of covert mortality nodavirus (CMNV) in

shrimp ponds. J Invertebr Pathol. 154:29-36.

Pooljun, C., Direkbusarakom, S., Chotipuntu, P., Hirono, I., Wuthisuthimethavee, S., 2016. Development of a TaqMan real-time RT-PCR

assay for detection of covert mortality nodavirus (CMNV) in penaeid shrimp. Aquaculture 464: 445–450.

Thitamadee, S., Prachumwat, A., Srisala, J., Jaroenlak, P., Salachan, P.V., Sritunyalucksana, K., Flegel, T.W., Itsathitphaisarn, O. 2016.

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Wang C., Liu, S., Li, X., Hao, J., Tang, K. and Zhang, Q., 2019. Infection of covert mortality nodavirus in Japanese flounder reveals host

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Wang C., Wang X.H., Liu S., Sang S.W. Zhang Q.L. 2018. Preliminary study on the natural infection of Carassius auratus with covert

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viral covert mortality disease (vcmd): disease card

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